A regulatory motif of fundamental importance to metabolic control is the allosteric modification of enzymatic activity by metabolites. The long term objective of this competitive renewal application continues to be to increase our understanding of the mechanisms by which allosteric ligands are able to modify enzymatic activity through binding to sites on an enzyme removed from the active site. In particular we are interested in systems in which the allosteric ligands achieve their effects by altering the affinity of the enzyme for its substrate. Three different allosteric enzymes will be studied: phosphofructokinase (PFK) from Escherichia coli, PFK from Bacillus stearothermophilus; and carbamoyl phosphate synthetase (CPS) from Escherichia coli. Each of these enzymes is now cloned and overexpressed in various E. coli strains so that copious quantities of enzyme are available for biophysical, thermodynamic, and kinetic studies. The strains also provide the means for generating site-directed mutants. By studying these enzymes in concert, a greater understanding of general properties exhibited by allosteric enzymes should be forthcoming than would result from a narrow focus on specific mechanistic issues presented by a single enzyme. Four new experimental approaches will be applied to the study of these enzymes: frequency-domain fluorescence spectroscopy, site-directed mutagenesis, isothermal microcalorimetry, and high hydrostatic pressure application. With these techniques the significance of the enthalpy and entropy contributions to the coupling free energy, which quantitatively defines both the nature and the magnitude of the allosteric effect, will be explored. In particular the question of why some enzymes seem to exhibit coupling free energies that are dominated by entropy changes for both activators and inhibitors, whereas for other enzymes coupling free energies are dominated by changes in enthalpy will be addressed. It is hypothesized that ligand-induced perturbations of the dynamics of the enzyme structure may contribute to the entropy component of the coupling free energy. If true, this hypothesis implies that much of what an allosteric ligand does upon binding might be invisible to structural depictions of enzyme-ligand complexes such as those afforded by x-ray crystallography.